Tyrosine-Dependent and -Independent Mechanisms of STAT3 Activation by the Human Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Are Differentially Utilized Depending on G-CSF Concentration

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Alister C. Ward ◽  
Mirjam H.A. Hermans ◽  
Louise Smith ◽  
Yvette M. van Aesch ◽  
Anita M. Schelen ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
Distal Region ◽  
Full Length ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stat Proteins ◽  
Jak2 Inhibitor ◽  
C Terminus ◽  
Necessary And Sufficient ◽  
Stimulating Factor

Abstract The granulocyte colony-stimulating factor receptor (G-CSF-R) activates multiple STAT proteins. Although the membrane-proximal cytoplasmic region of the G-CSF-R is necessary and sufficient for activation of STAT1 and STAT5, activation of STAT3 requires the membrane distal region that contains four tyrosines. Although one of these (Y704) has previously been shown to be involved in STAT3 activation from a truncated G-CSF-R derived from a patient with severe chronic neutropenia (SCN), this tyrosine is not required for STAT3 activation by the full-length G-CSF-R. To investigate possible alternative mechanisms of STAT3 activation, we generated a series of Ba/F3 cell transfectants expressing the wild-type G-CSF-R or mutant receptors that either completely lack tyrosines or retain just one of the four cytoplasmic tyrosines of the G-CSF-R. We show that, at saturating G-CSF concentrations, STAT3 activation from the full-length G-CSF-R is efficiently mediated by the C-terminal domain in a manner independent of receptor tyrosines. In contrast, at low G-CSF concentrations, Y704 and Y744 of the G-CSF-R play a major role in STAT3 activation. Both tyrosine-dependent and -independent mechanisms of STAT3 activation are sensitive to the Jak2 inhibitor AG-490, follow similar kinetics, and lead to transactivation of a STAT3 reporter construct, indicating functional equivalence. STAT3 activation is also impaired, particularly at nonsaturating G-CSF concentrations, in bone marrow cells from mice expressing a truncated G-CSF-R (gcsfr-▵715). These findings suggest that G-CSF–induced STAT3 activation during basal granulopoiesis (low G-CSF) and “emergency” granulopoiesis (high G-CSF) are differentially controlled. In addition, the data establish the importance of the G-CSF-R C-terminus in STAT3 activation in primary cells, which has implications for understanding why truncated G-CSF-R derived from SCN patients are defective in maturation signaling.

Tyrosine-Dependent and -Independent Mechanisms of STAT3 Activation by the Human Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Are Differentially Utilized Depending on G-CSF Concentration

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Alister C. Ward ◽  
Mirjam H.A. Hermans ◽  
Louise Smith ◽  
Yvette M. van Aesch ◽  
Anita M. Schelen ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
Distal Region ◽  
Full Length ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stat Proteins ◽  
Jak2 Inhibitor ◽  
C Terminus ◽  
Necessary And Sufficient ◽  
Stimulating Factor

The granulocyte colony-stimulating factor receptor (G-CSF-R) activates multiple STAT proteins. Although the membrane-proximal cytoplasmic region of the G-CSF-R is necessary and sufficient for activation of STAT1 and STAT5, activation of STAT3 requires the membrane distal region that contains four tyrosines. Although one of these (Y704) has previously been shown to be involved in STAT3 activation from a truncated G-CSF-R derived from a patient with severe chronic neutropenia (SCN), this tyrosine is not required for STAT3 activation by the full-length G-CSF-R. To investigate possible alternative mechanisms of STAT3 activation, we generated a series of Ba/F3 cell transfectants expressing the wild-type G-CSF-R or mutant receptors that either completely lack tyrosines or retain just one of the four cytoplasmic tyrosines of the G-CSF-R. We show that, at saturating G-CSF concentrations, STAT3 activation from the full-length G-CSF-R is efficiently mediated by the C-terminal domain in a manner independent of receptor tyrosines. In contrast, at low G-CSF concentrations, Y704 and Y744 of the G-CSF-R play a major role in STAT3 activation. Both tyrosine-dependent and -independent mechanisms of STAT3 activation are sensitive to the Jak2 inhibitor AG-490, follow similar kinetics, and lead to transactivation of a STAT3 reporter construct, indicating functional equivalence. STAT3 activation is also impaired, particularly at nonsaturating G-CSF concentrations, in bone marrow cells from mice expressing a truncated G-CSF-R (gcsfr-▵715). These findings suggest that G-CSF–induced STAT3 activation during basal granulopoiesis (low G-CSF) and “emergency” granulopoiesis (high G-CSF) are differentially controlled. In addition, the data establish the importance of the G-CSF-R C-terminus in STAT3 activation in primary cells, which has implications for understanding why truncated G-CSF-R derived from SCN patients are defective in maturation signaling.

scholarly journals Granulocyte colony-stimulating factor activation of Stat3 alpha and Stat3 beta in immature normal and leukemic human myeloid cells

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2442-2449 ◽  
Author(s):  
A Chakraborty ◽  
SM White ◽  
TS Schaefer ◽  
ED Ball ◽  
KF Dyer ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
Short Form ◽  
Gene Activation ◽  
Myeloid Cells ◽  
Myelogenous Leukemia ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stat Proteins ◽  
Human Cd34 ◽  
Stimulating Factor

Granulocyte colony-stimulating factor (G-CSF) is the cytokine critical for directing neutrophilic granulocyte differentiation. Acute myelogenous leukemia (AML) cells, which frequently arise from this lineage, respond aberrantly to G-CSF by proliferating without differentiating. The basis for this abnormal responses is unknown. In the present study, we investigated whether G-CSF signaling in immature normal and leukemic human myeloid cells diverges at the level of activation of signal transducers and activators of transcription (STAT) proteins. We compared the profile of STAT proteins activated in G-CSF-stimulated immature normal and leukemic human myeloid cells. G-CSF activated Stat3 alpha in all AML cell lines examined except HL60 and in three of six uncultured AML patient samples. In normal human CD34+ bone marrow cells and HL60 cells, both reported to differentiate in response to G-CSF, G-CSF did not activate Stat3 alpha; rather, it activated only an 83 kD form of Stat3 that proved to be the human homologue of a short form of Stat3, Stat3 beta. Because the transcriptional activity of Stat3 beta is distinct from Stat3 alpha, these results suggest that the balance of the two Stat3 isoforms in myeloid cells may influence the cellular pattern of gene activation and consequently the ability of these cells to differentiate in response to G-CSF.

scholarly journals Essential Role for Cyclin D3 in Granulocyte Colony-Stimulating Factor-Driven Expansion of Neutrophil Granulocytes

2006 ◽  
Vol 26 (21) ◽  
pp. 8052-8060 ◽  
Author(s):  
Ewa Sicinska ◽  
Young-Mi Lee ◽  
Judith Gits ◽  
Hirokazu Shigematsu ◽  
Qunyan Yu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Cyclin D3 ◽  
Neutrophil Granulocytes ◽  
Neutrophil Granulocyte ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Stimulating Factor

ABSTRACT The proliferation of neutrophil granulocyte lineage is driven largely by granulocyte colony-stimulating factor (G-CSF) acting via the G-CSF receptors. In this study, we show that mice lacking cyclin D3, a component of the core cell cycle machinery, are refractory to stimulation by the G-CSF. Consequently, cyclin D3-null mice display deficient maturation of granulocytes in the bone marrow and have reduced levels of neutrophil granulocytes in their peripheral blood. The mutant mice are unable to mount a normal response to bacterial challenge and succumb to microbial infections. In contrast, the expansion of hematopoietic stem cells and lineage-committed myeloid progenitors proceeds relatively normally in mice lacking cyclin D3, revealing that the requirement for cyclin D3 function operates at later stages of neutrophil development. Importantly, we verified that this requirement is specific to cyclin D3, as mice lacking other G1 cyclins (D1, D2, E1, or E2) display normal granulocyte counts. Our analyses revealed that in the bone marrow cells of wild-type mice, activation of the G-CSF receptor leads to upregulation of cyclin D3. Collectively, these results demonstrate that cyclin D3 is an essential cell cycle recipient of G-CSF signaling, and they provide a molecular link of how G-CSF-dependent signaling triggers cell proliferation.

scholarly journals Target cells for granulocyte colony-stimulating factor, interleukin-3, and interleukin-5 in differentiation pathways of neutrophils and eosinophils

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 1956-1961 ◽  
Author(s):  
H Ema ◽  
T Suda ◽  
K Nagayoshi ◽  
Y Miura ◽  
CI Civin ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
Early Stage ◽  
Target Cells ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Cd34 Antigen ◽  
Cd34 Cells ◽  
Stimulating Factor

Abstract To study the relationship between hematopoietic factors and their responsive hematopoietic progenitors in the differentiation process, both purified factors and enriched progenitors are required. We isolated total CD34+ cells, CD34+,CD33+ cells, and CD34+,CD33- cells individually from normal human bone marrow cells by fluorescence- activated cell sorter (FACS), and examined the effects of granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), and IL-5 on in vitro colony formation of these cells. CD34+,CD33+ cells formed granulocyte colonies in the presence of G-CSF. Both CD34+,CD33+ cells and CD34+,CD33- cells formed granulocyte/macrophage colonies in the presence of IL-3. Eosinophil (Eo) colonies were only formed by CD34+,CD33- cells in response to IL-3, but scarcely formed by CD34+ cells in the presence of IL-5. We performed the two-step cultures consisting of the primary liquid culture for 6 days and the secondary methylcellulose culture, and serially examined changes in phenotypes of ,he cells cultured in the primary culture. CD34-,CD33+ cells derived from CD34+,CD33+ cells by preincubation with G-CSF or IL-3 formed Eo colonies in the presence of IL-5 but not IL-3. CD34-,CD33+ cells derived from CD34+,CD33- cells by preincubation with IL-3 also formed Eo colonies by support of IL-5 as well as IL-3. Both CD34+ cells gradually lost the CD34 antigen by day 6 of incubation with G-CSF or IL- 3. Loss of this antigen was well-correlated with acquisition of susceptibility to IL-5. It was concluded that G-CSF supported the neutrophil differentiation of committed colony-forming cells, IL-3 supported that of both committed and multipotent colony-forming cells. G-CSF and IL-3 also supported the early stage of E. differentiation; IL- 5 supported the late stage of that.

Increased granulopoiesis through interleukin-17 and granulocyte colony-stimulating factor in leukocyte adhesion molecule–deficient mice

Blood ◽  
2001 ◽  
Vol 98 (12) ◽  
pp. 3309-3314 ◽  
Author(s):  
S. Bradley Forlow ◽  
Jill R. Schurr ◽  
Jay K. Kolls ◽  
Gregory J. Bagby ◽  
Paul O. Schwarzenberger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Feedback Loop ◽  
Bone Marrow Cells ◽  
Leukocyte Adhesion ◽  
Interleukin 17 ◽  
Blood Neutrophil ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Peripheral Blood Neutrophil ◽  
Stimulating Factor

Abstract Many mutant mice deficient in leukocyte adhesion molecules display altered hematopoiesis and neutrophilia. This study investigated whether peripheral blood neutrophil concentrations in these mice are elevated as a result of accumulation of neutrophils in the circulation or altered hematopoiesis mediated by a disrupted regulatory feedback loop. Chimeric mice were generated by transplanting various ratios of CD18+/+ and CD18−/− unfractionated bone marrow cells into lethally irradiated wild-type mice, resulting in approximately 0%, 10%, 50%, 90%, or 100% CD18 null neutrophils in the blood. The presence of only 10% CD18+/+ neutrophils was sufficient to prevent the severe neutrophilia seen in mice reconstituted with CD18−/− bone marrow cells. These data show that the neutrophilia in CD18−/− mice is not caused by enhanced neutrophil survival or the inability of neutrophils to leave the vascular compartment. In CD18−/−, CD18−/−E−/−, CD18−/−P−/−, EP−/−, and EPI−/− mice, levels of granulocyte colony-stimulating factor (G-CSF) and interleukin-17 (IL-17) were elevated in proportion to the neutrophilia seen in these mice, regardless of the underlying mutation. Antibiotic treatment or the propensity to develop skin lesions did not correlate with neutrophil counts. Blocking IL-17 or G-CSF function in vivo significantly reduced neutrophil counts in severely neutrophilic mice by approximately 50% (P < .05) or 70% (P < .01), respectively. These data show that peripheral blood neutrophil numbers are regulated by a feedback loop involving G-CSF and IL-17 and that this feedback loop is disrupted when neutrophils cannot migrate into peripheral tissues.

scholarly journals Proliferation Signaling and Activation of Shc, p21Ras, and Myc Via Tyrosine 764 of Human Granulocyte Colony-Stimulating Factor Receptor

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1924-1933 ◽  
Author(s):  
John P. de Koning ◽  
Amrita A. Soede-Bobok ◽  
Anita M. Schelen ◽  
Louise Smith ◽  
Daphne van Leeuwen ◽  
...  
Keyword(s):  
Distal Region ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Signaling Mechanisms ◽  
Fold Reduction ◽  
Multiple Signaling ◽  
Stimulating Factor

The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF–mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF–containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF–mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF–driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.

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